Phosphorothioate Oligonucleotides as Microbicides against HIV Transmission

描述（由申请人提供）：制定抑制艾滋病毒感染传播的干预措施对于制止艾滋病毒流行至关重要。局部预防战略通常被称为杀微生物剂，已被提议作为阻止或减缓艾滋病毒流行的一种战略。我们已经鉴定出新型铅杀菌剂，在体外有效抑制HIV和SIV感染/复制。在我们之前的提交中，我们报道了一个具有磷酸化骨架（OPB）的寡核苷酸，可以分别抑制HIVBaL或SIVmac251在人类或类人猿PBMC中的感染和/或复制。OPB还能抑制HIVBaL和HIVIIIB对P4-R5 MAGI细胞无细胞感染的感染/复制。连续暴露24小时后，OPB对PBMC和P4-R5 MAGI细胞无毒性作用。初步数据显示，OPB对其他病毒也有抑制作用，因为它对甲型流感病毒也有效。因此，我们的第一代OPB可能是一种有效的抗HIV杀菌剂，局部应用时可以防止粘膜部位的感染。我们对OPB的聚T或聚a寡核苷酸进行了初步研究，这表明这种作用与序列无关，甚至可能由磷酸硫代脱氧核糖主干介导。事实上，在我们目前的重新提交中，我们展示了我们的下一代化合物的数据，这是一种无基硫代2'脱氧核糖骨架（PDB），具有比OPB更有效的HIV抑制活性。我们在这里展示的一种14聚PDB没有毒性，是一种有效的HIV抑制剂，并且具有作为TLR7/9拮抗剂抑制HIV诱导的IFN¿产生的优势。后一种特性很重要，因为HIV感染的建立可能取决于TLR引发的HIV诱导的粘膜炎症。重要的是，我们表明PDB在pH 4.4下在羟乙基纤维素（HEC）凝胶中配制时具有活性，可以在pH过渡到中性pH时存活，并且在HEC中长时间保持活性。我们假设PDB结合包膜病毒并通过作为“化学凝集素”抑制其传染性。我们进一步假设PDB可以作为抗HIV的杀微生物剂，并可以预防恒河猴SIV阴道感染。R21期计划开展的研究将进一步优化和表征PDB体外安全性和有效性，以及其在Swiss Webster小鼠阴道/宫颈刺激模型中的安全性。他们将确定最佳的大小和组成，仍然有效地对抗艾滋病毒，并表现出无毒。最后，研究PDB的作用机理，测试其在羟乙基纤维素凝胶中的包合效果，确定PDB对共生乳酸菌生长的影响。从R21阶段到R33阶段，已经设定了五个具体的里程碑。R33期将测试PDB预防阴道SIV感染的有效性，研究精浆和pH变化对OPB疗效的影响，确定其与人生殖器上皮组织的安全性，并研究其对HSV-2的有效性。目前的应用将允许广泛评估PDB作为可能的新型杀微生物剂候选物。这里提出的研究解决了重要的公共卫生问题，即开发抑制艾滋病毒感染传播的治疗方法。目前的申请是研究一种可能用于抑制HIV感染的新型化学物质。

DESCRIPTION (provided by applicant): Developing interventions that inhibit the transmission of HIV infection are critical for halting the HIV epidemic. Topical prevention strategies usually termed microbicides have been proposed as one strategy to halt or slow down the HIV epidemic. We have identified novel lead microbicides that potently inhibit HIV and SIV infection/replication in vitro. During our previous submission we reported an oligonucleotide with a phosphorothioate backbone (OPB) that could inhibit HIVBaL or SIVmac251 infection and/or replication in human or simian PBMC, respectively. OPB also inhibited infection/replication in cell-free infections of P4-R5 MAGI cells by HIVBaL and HIVIIIB. OPB exhibited no toxicity against PBMC or P4-R5 MAGI cells after 24h continuous exposure. Preliminary data suggested that OPB may also inhibit other viruses as it was also effective against influenza type A virus. Thus, our first generation OPB may be a potent microbicide against HIV that prevents infection at mucosal sites when topically applied. Our preliminary studies were carried out with a 13mer Poly T or Poly A oligonucleotide of OPB and this suggested that the effect was sequence independent and may even be mediated by the phosphorothioate deoxyribose sugar backbone. Indeed in our current re-submission we present data on our next generation compound, a baseless phosphorothioate 2' deoxyribose backbone (PDB) that has more potent HIV inhibitory activity than OPB. A 14mer PDB we show here has no toxicity, is a potent inhibitor of HIV and has the advantage of being a TLR7/9 antagonist that inhibits HIV-induced IFN¿ production. This later property is important as the establishment of HIV infection may depend on HIV-induced mucosal inflammation triggered by TLR. Importantly, we show that PDB is active when formulated in hydroxyethylcellulose (HEC) gel at pH 4.4, survives pH transition to a neutral pH, and in retains its activity in HEC for long periods. We hypothesize that PDB binds enveloped viruses and inhibits their infectivity by acting as a "chemical lectin". We further hypothesize that PDB can act as a microbicide against HIV and can prevent SIV vaginal infection of rhesus macaques. The studies planned in the R21 phase will further optimize and characterize the safety and effectiveness of PDB in vitro and its safety in the Swiss Webster mouse vaginal/cervical model of irritation. They will determine the optimal size and composition that remains effective against HIV and exhibits no toxicity. Finally, the mechanism of action of PDB will be investigated, the effect of inclusion into hydroxyethylcellulose gel will be tested and PDB's effect on the growth of commensal lactobacilli will be determined. Five specific milestones have been set for the progression from the R21 Phase to the R33 Phase. The R33 phase will test the effectiveness of PDB in preventing vaginal SIV infection, investigate the effect of seminal plasma and pH transition on the efficacy of OPB, determine its safety with human genital epithelial tissue, and investigate its effectiveness against HSV-2. The current application will allow for an extensive evaluation of PDB as possible novel microbicide candidates. The studies proposed here address the important public health problem of developing treatments that inhibit the transmission of HIV infection. The current application investigates a novel chemical that may be used to inhibit infection with HIV.